Dynamics of Regulatory RNAs in Bacteria Small non-coding RNAs (sRNAs) in bacteria regulate cell growth and stress response, coordinating the levels of gene expression in response to diverse environmental conditions. Genes regulated by sRNAs control host interactions, virulence and toxin expression in many pathogenic bacteria, and thus have a direct impact on human health. How sRNAs recognize and base pair with their mRNA targets is not understood, in part because each RNA molecule must change structure in order for the two RNAs to pair with each other. Base pairing between sRNAs and mRNAs is facilitated by a protein chaperone Hfq, which is required for genetic regulation by sRNAs. The physical interactions between sRNAs, mRNAs and Hfq protein dictate how these regulatory switches operate and how different regulatory pathways intersect within the same bacterium. Biochemical, physical and genetic methods will used to study how bacterial non-coding sRNAs control gene expression, using the master stress-response regulator rpoS as a model. The 3D conformation of the Hfq-rpoS RNA complex will be determined by biochemical footprinting, mutagenesis and SAXS. The mechanism by which Hfq recruits sRNAs and stimulates pairing with mRNA targets will be investigated using fluorescence spectroscopy and protein engineering. The requirement for Hfq binding sites in mRNAs and sRNAs in vivo will be compared in negatively and positively regulated mRNAs, using rpoS-lacZ translational fusions in E. coli. Results of genetic experiments will be correlated with in vitro measurements of sRNA annealing and Hfq binding. The results will show how the structure or stability of Hfq-sRNA- mRNA complexes determines downstream regulatory outcomes, which is currently unknown. As Hfq is homologous to eukaryotic Sm proteins, the results will be also relevant to understanding how Sm proteins act in human mRNA turnover and pre-mRNA splicing. PUBLIC HEALTH RELEVANCE: PROJECT DESCRIPTION Bacterial pathogens use small RNA molecules to control genes that help them survive in the human body and produce toxins after infection. The goal of this research project is to determine the physical shapes of these small RNAs, and how they come together with messenger RNAs and a RNA chaperone protein to turn genes on and off in bacterial cells.